This invention relates to an oligosaccharide derivative and a process for measuring .alpha.-amylase activity by using the oligosaccharide derivative as a substrate.
Measurement of .alpha.-amylase activity in a sample, particularly in saliva, pancreatic juice, blood and urine in human living body is important for diagnosis in medical science. For example, .alpha.-amylase activity in blood and urine shows a remarkable increase in the case of pancreatitis, cancer of the pancreas, and parotitis compared with normal values.
Various methods for measuring .alpha.-amylase activity have been reported. These methods can be divided into two groups, one of which is to use a long chain natural product such as starch, amylose, amylopectin, or the like or a modified material thereof as a substrate, and another of which is to use an oligosaccharide having 4 to 7 glucose units or a derivative thereof as a substrate.
Recently, methods of using uniform substances having definite structures as a substrate are to be employed widely in place of known methods using starch as a substrate. For example, there are proposed a method of using an oligosaccharide such as maltotetraose (G.sub.4), maltopentaose (G.sub.5), maltohexaose (G.sub.6), or maltoheptaose (G.sub.7) as a substrate (Chem. Abstr. 82, 151408 h (1975); ibid. 88, 148201 d (1978)), a method of using an oligosaccharide bonding a chromogen such as p-nitrophenol at a reducing end thereof (Chem. Abstr., 90, 182383 r (1979)), etc.
These methods generally require such a coupling enzyme for the measurement as .alpha.-glucosidase (E.C. 3.2.1.20: .alpha.-D-glucoside glucohydrolase), glucoamylase (E.C. 3.2.1.3: 1,4-.alpha.-D-glucan glucohydrolase), or .beta.-glucosidase (E.C. 3.2.1.21: .beta.-D-glucoside glucohydrolase). Since these coupling enzymes are exo type enzymes which hydrolyze an .alpha.-1,4-glucoside bond from a non-reducing end of saccharide chain having .alpha.-1,4-glucoside bonds, they have a defect in that they decompose the substrates irrespective of the .alpha.-amylase reaction. Therefore, in the above-mentioned measuring methods using these coupling enzymes, a reagent solution for the measurement becomes unstable, and reagent blank values become remarkably high, which results in making measuring accuracy remarkably worse. Further, since a sufficient amount of glucoamylase or .alpha.-glucosidase necessary for the measurement cannot be used, it is difficult to construct a measuring method with high accuracy.
In order to solve such problems, the present inventors have synthesized modified oligosaccharides represented by the formula: ##STR2## wherein the rightmost glucose unit is a reducing group; k is an integer of 2 to 5; and R is an organic residue such as a pyridylamino group, and used them as a substrate for measuring .alpha.-amylase activity (European Patent Publication No. 0104047 A2). These substances are uniform and definite in structure as the substrate and characterized by not becoming a substrate for .alpha.-glucosidase, .beta.-glucosidase or glucoamylase. When these substances are used as a substrate for measuring .alpha.-amylase activity, there must employ either a high performance liquid chromatographic method or a method wherein a coupling enzyme such as .alpha.-glucosidase, .beta.-glucosidase or glucoamylase is used to produce glucose which is subjected to the measurement. Therefore, this method has problems in that a special device should be used in the former case and an influence of glucose contained in a sample cannot be neglected in the latter case, and the like.
On the other hand, Chem. Abstr., 102, 221146d (1985) discloses an oligoglucoside derivative of the formula: ##STR3## wherein R and R.sub.1 are independently a straight-chain or branched alkyl or alkoxy group having 1 to 6 carbon atoms or a phenyl group, R and R.sub.1 in combination being able to form a methylene bridge and at least one of hydrogen atoms may independently be substituted with an alkyl group having 1 to 5 carbon atoms or a phenyl group; R.sub.2 is a glucoside group having 2 to 7 glucose units; and X is hydrogen or a group which can be measured optically, particularly a nitrophenyl group, and a method for measuring .alpha.-amylase activity by using such an oligoglucoside derivative as a substrate. But when R and R.sub.1 are alkyl, alkoxy or phenyl groups, it is difficult to synthesize the compound of the formula (II) in good yield (due to the introduction of these groups into the hydroxyl groups at the 2- and 3-positions, difficulty in isolation and poor yield). On the other hand, when R and R.sub.1 together form a methylene bridge, the resulting ethylidene type compound of the formula (II) is unstable (even at neutral) when used as a substrate. Further, the compound of the formula (II) has some problems in that the efficiency of this compound as a substrate is poor.
As mentioned above, substrates heretofore known are not satisfactory and require further improvement.